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Nanotoxicology
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Drosophila
as a model for assessing nanopesticide
toxicity
Eşref Demir
To cite this article: Eşref Demir (2020) Drosophila as a model for assessing nanopesticide toxicity, Nanotoxicology, 14:9, 1271-1279, DOI: 10.1080/17435390.2020.1815886
To link to this article: https://doi.org/10.1080/17435390.2020.1815886
Published online: 24 Sep 2020.
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ORIGINAL ARTICLE
Drosophila as a model for assessing nanopesticide toxicity
Es¸ref Demir
Department of Medical Services and Techniques, Medical Laboratory Techniques Programme, Antalya Bilim University, Vocational School of Health Services, Antalya, Turkey
ABSTRACT
One of the fastest-moving fields in today’s world of applied science, nanotechnology allows the control and design of matter on an extremely small scale, so it has now become an integral part of various industries and scientific areas, such as agriculture, food sector, healthcare and engineering. Understanding the interactions between nanopesticides and edible plants, as well as non-target animals, is crucial in assessing the potential impact of nanotechnology products on the environment, agriculture and human health. The dramatic increase in efforts to use nanopesticides renders the risk assessment of their toxicity and genotoxicity highly crucial due to the potential adverse impact of this relatively uncharted territory. Such widespread use natur-ally increases our exposure to nanopesticides, raising concerns over their possible adverse effects on humans and non-target organisms, which might include severe impairment of both male and female reproductive capacity. We therefore need better insight into such effects to derive conclusive evidence on the safety or toxicity/genotoxicity of nanopesticides, and Drosophila melanogaster (fruit fly) can prove an ideal model organism for the risk assessment and toxicological classification of nanopesticides, as it bears striking similarities to various sys-tems in human body. This editorial review attempts to summarize our current knowledge derived from previous in vivo studies to examine the impact of several nanomaterials on various species of mammals and non-target model organisms at the genetic, cellular, and molecular lev-els, attracting attention to the possible mechanisms and potential toxic/genotoxic effects of nanopesticides widely used in agriculture on D. melanogaster as a non-target organism.
ARTICLE HISTORY Received 1 July 2020 Revised 21 August 2020 Accepted 23 August 2020 KEYWORDS Nanopesticides; Drosophila melanogaster; in vivo model organism; risk assessment; environment and health
Issue with pesticides
The use of pesticides in agriculture, either from organic or synthetic sources, serves many crucial functions, such as securing crop yield against inva-sive organisms and enabling farmers to produce safe and quality foods at affordable prices. Without pesti-cide use, more than 50% of the global agricultural output would be lost to diseases and pests (OECD-FAO Agricultural Outlook 2012). However, conven-tional pesticides bring about some serious environ-mental drawbacks. For instance, most tend to be nonspecific, and thus, along with invasive species, also damage harmless or even beneficial organisms like bees. Indeed, it has been estimated that only about 0.1% of the pesticides reach the target species during aerial spraying, with the rest polluting the surrounding ecosystem (Carriger et al. 2006). Besides, pesticides are also known to contaminate soil and water resources, and although we have
limited evidence to measure the total health impact of pesticides across the globe, they have been reported to cause about 100 000 human deaths worldwide are due to acute and chronic poisoning (WHO Global Health Statistics 2015), with children and women in developing countries being particu-larly vulnerable to the toxic effects of pesticides.
The aims of this review are to present a compre-hensive overview of all apparent studies carried out with nanopesticides and Drosophila melanogaster, to attain a clear and comprehensive picture of the potential risk of nanopesticide exposure to health, and to demonstrate the advantages of using Drosophila with new technology (for example CRISPR/Cas9 for pesticide resistance) in this field.
Nanopesticides as a solution
In recent years, the development and application of nanomaterial-based formulations in pesticide
CONTACTEs¸ref Demir esref.demir@antalya.edu.tr Department of Medical Services and Techniques, Medical Laboratory Techniques Programme, Antalya Bilim University, Vocational School of Health Services, Antalya, Turkey
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2020, VOL. 14, NO. 9, 1271–1279
production has emerged as a potential solution to the unwanted effects of conventional pesticides. Nanopesticides include nanomaterials (NMs) used as carriers of pesticidal substances to enable their con-trolled release at more efficient doses and refer to a wide range of products combining various surfac-tants, capsules, metal oxides, particles, and poly-mers on the nanoscale (often measuring 1–100 Nm) (Table 1). These include nano form of pyrifluquinaz, which is designed to modify insect behavior by interfering with the insect’s feeding activity (Kang et al. 2012), nanocapsules of botanical insecticides [(the major constituents of Eucalyptus extract: 1,8-cineole (70.94%) and 1,2-benzenedicarboxylic acid (6.08%)] (Khoshrafta et al. 2019), nano-size silver (Ag) colloidal solution as fungicidal (Kim et al. 2012), neem-oil loaded zein nanoparticles (NPs) (Pascoli et al. 2019), fungus Chrysosporium tropi-cum used for synthesizing the Ag and gold (Au) NPs as a larvicide against Aedes aegypti (Soni and Prakash 2012), Mesoporous silica NPs (MSN) for stor-age and controlled release of metalaxyl fungicide (Wanyika 2013). Another example is a nanogel pro-duced from methyl eugenol (a pheromone) using a low-molecular mass gelator to prevent pests from
harming a range of fruits such as guava (Bhagat, Samanta, and Bhattacharya2013).
This editorial review aims to present a concise overview of previous studies that examine possible adverse effects of different nanomaterials on non-target model organisms, to attract attention to the potential mechanisms and toxic/genotoxic effects of nanopesticides widely used in agriculture on Drosophila as a non-target model organism, and to demonstrate the advantages of using it with new technology [such as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)] for pesticide resistance.
Unlike conventional pesticides, formulations con-taining NMs as additives are often designed to improve solubility of the active ingredient (AI) and to release the chemical in a precise and efficient way, thus protecting it from early degradation (Kookana et al. 2014). Nanoparticles (NPs) can reduce the required amount of pesticide for pest control by enhancing the durability and efficacy of chemicals. Nanopesticides may also contain ultra-fine particles of AI, such as Ag, Au, and titanium dioxide (TiO2) that are toxic to pests (Bergeson 2010; Kah and Hofmann 2014; Sekhon 2014). For
Table 1. Categories of nanopesticides.
Type Nanotechnology Advantages
AI (Active Ingredient) Target Pest References Nanoclays NPs of layered mineral silicates
Efficient carriers of pesticides Controlled release of AI High absorption potential Better insecticidal activity
than microparticles Cypermethrin (insecticide) Metalaxyl (fungicide) Mosquitoes Fleas Spiders Termites Fungi Wanyika2013 Xiang et al.2017 Nanocapsules Polymeric NPs or nanoscale shells made from polymers to encapsulate AI
Efficient uptake and effectiveness against pests Efficient targeting
Sustained pesticide release
Eucalyptus globulus extract Garlic oil Thymus herba-barona Greenfly Flour beetle Whitefly Khoshraftar et al.2019
Nanoemulsions AI loaded into nanoscale oil droplets
High cellular uptake by organisms
Improved AI distribution Low toxicity to
non-target organisms
Neem oil (from Azadirachta indica tree) Permethrin Whitefly Aphids Moth larvae Spider mites Yellow fewer mosquito
Mishra et al.2018
Pascoli et al.2019
Nanogels Natural or synthetic polymers with a diameter of 10-100 nm. The pores in the gel can be loaded with AI
Long lasting residual activity Increased efficacy
Long shelf life Low-cost Good safety profile
Methyl eugenol Oriental fruit fly (Bactrocera dorsalis)
Bhagat, Samanta, and
Bhattacharya
2013
Nanoliposomes They are structures at nanoscale used for the encapsulation and delivery of AI High biocompatibility Biodegradable Improved stability of sensitive materials Sustained release of AI
Clove oil (eugenol) Etofenprox Pyrifluquinazon
Mosquitoes Leaf hoppers Whitefly Rice water weevils
Hwang et al.2011
Kang et al.2012
Inorganic NPs Elemental metals, metal oxides, and metal salts used as
nanopesticide AI
High effectiveness with increased toxicity to pests Excellent AI distribution Highly stable as compared to
organic NMs Gold NPs Titanium dioxide NPs Silver NPs Copper NPs Aedes aegypti Fungi Bacteria
Soni and Prakash
2012Kim et al.2012
example, Ag NPs, known to have antimicrobial properties (Kim et al. 2012), have been successfully used as active ingredients against harmful plant pathogens such as rice blast fungus (Magnaporthe grisea). Thanks to today’s advanced nanotechnol-ogy, NPs can be modified by altering their size, shape, surface area, and surface charge of particles to make them more specific to target organisms as compared to conventional pesticides, insecticides, and insect repellents (Sasson et al. 2007). For example, nanocapsules can slow the release of sub-stance in liquid or solid forms to specific plants through release mechanisms such as dissolution, biodegradation, diffusion, and osmotic pressure (Vidyalakshmi, Bhakyaraj, and Subhasree 2009). Nanogels containing small pores loaded with the pheromone methyl eugenol can be applied to a number of fruit crops as a protection against Bactrocera dorsalis. They have been found to pre-serve the active ingredient during the whole period of insect growth, thus allowing an effective pest control (Bhagat, Samanta, and Bhattacharya 2013). Furthermore, Polyethylene glycol (PEG) coated NPs loaded with garlic essential oil have been reported to be highly effective against harmful pests like Tribolium castaneum Herbst (Yang et al. 2009). Finally, silica NPs (Si NPs) are known to improve plant tolerance to biotic stresses, and amorphous nanosilica has been used for agricultural pest con-trol (Barik, Sahu, and Swain2008).
Production of nanopesticides involves the use of NMs with pesticidal properties like Ag, Au, and TiO2
NPs as active ingredients or as nanocapsules to improve delivery systems. In both cases, the main routes of exposure to nanopesticides are via inhal-ation, ingestion of the foods containing nanopesti-cides, and dermal contact. Considering the higher cellular uptake of NPs once they have reached sev-eral systems in the organism, as compared to bulk materials in conventional pesticides, they may prove even more toxic to non-target organisms that play a crucial role in the ecosystem, which might include common pollinators like honey bees and birds. Such an effect could indirectly cause a signifi-cant loss of crop yield due to insufficient population of pollinators (Fishel 2011). For that reason, future production of bioactive agent-based nanopesticides containing silver and other materials should take into account the correct size and concentration of
NPs that would be effective against pests and inva-sive pathogens but less toxic to non-target organ-isms. Therefore, a detailed investigation of interactions between engineered NMs and living systems seems highly crucial to develop pesticidal nanoformulations that can modulate specific molecular pathways.
Altogether, nanopesticides have emerged as a potential solution to address the toxic effects of pesticides. However, before they can be used for agricultural application, we need to better under-stand their impact on non-target animal species and ecosystem (Kah et al.2013).
Addressing the toxicity of nanopesticides
The recent interest toward the use of nanopesti-cides in agriculture raises concerns over their pos-sible toxic/genotoxic impacts on humans as well as non-target organisms, Thus, effective risk assess-ment tools are needed to evaluate their biological interactions, ecotoxicity, and cytotoxicity. The risk assessment of pesticide product formulations (PPFs) have previously been performed by evaluating the toxicity of the active ingredient, neglecting the potential hazards of other components or possible combined effects of mixtures. Thus, it is imperative to assess the toxicity of various nanopesticide for-mulations more systematically to fully understand the risks associated with their use. In particular, interactions of NMs and coating materials should be examined (Nagy et al.2020).
Unlike NMs, larger bulk materials exhibit certain physical properties independent of their size, but at the nanoscale such properties may show dramatic variations depending on the particle size and shape. This especially applies to metal oxide NPs that have a great surface area, since it plays a key role in its reactivity and other physicochemical properties (Golbamaki et al. 2015). NMs can be engineered to impart special functions such as enhanced strength, magnetic, thermal and electrical conductivity and also to produce structures with high surface-to-vol-ume ratios (Sanvicens and Marco 2008; Sau et al. 2010). Such novel functionalities and properties may result in unexpected biological and chemical reactivity, increasing their toxicity to humans and animals. Features such as particle size, surface area, shape, and surface coating appear to regulate
cytotoxicity of NPs (Macaroff et al. 2006). For example, small particles are known to be more toxic to living cells than large ones (Gliga et al. 2014), suggesting that NPs with large surface-to-volume ratio may be more toxic (Donaldson et al. 2004). Thus, it is crucial to fully assess the possible toxicity of NPs, which include cell toxicity and DNA damage (Snyder-Talkington et al. 2012). Although in vitro testing using various cell types yield valuable data, the use of tissue culture approaches often fail to reflect what really happens within a living organism upon exposure to specific materials (Lewinski, Colvin, and Drezek 2008). Therefore, researchers tend to conduct in vivo nanotoxicology studies often using target organisms (pests) to test poten-tial effects of NMs like Ag NPs and Au NPs used in pest control products. For example, an in vivo study using Ag NPs and Au NPs as active ingredients tested their pesticidal capacity on the yellow fever mosquito (Aedes aegypti) (Soni and Prakash 2012; Kim et al. 2012). Exposure to NPs exerted highly toxic effects on larvae, with mortality rates as high as 100%, leading to the conclusion that the use of Au and Ag NPs could be an effective choice for safer and environmentally friendlier mosquito con-trol (Soni and Prakash2012). However, pesticide for-mulations containing NMs as additives or active ingredients should also be thoroughly tested for their possible toxic effects on non-target animal species that are harmless or beneficial to nature and humans, including in particular pollinators like bees and predators hunting insects categorized as pests. Considering that only 1% of all insects are considered as pests (Triplehorn and Johnson 2005), nanopesticides could cause serious damage to inte-gral parts of the ecosystem by destroying benign organisms that regulate the population of invasive species and pests.
Drosophila as a model for assessing the toxicity of nanopesticides
A thorough investigation of nanopesticides formula-tions for possible toxic effects on in vivo animal models is critical. Several factors such as high oper-ational costs and ethical issues tend to restrict the use of traditional in vivo testing (mammalian acute toxicity testing), thus simpler experimental models like roundworms, zebrafish, and fruit flies. Among
them, Drosophila melanogaster stands out as an ideal in vivo model organism for assessing the cyto-toxicity and genotoxicity of nanopesticides. Drosophila, a member of the family Drosophilidae, has for some time attracted serious scholarly atten-tion and gained acceptance across diverse fields of biological and medical research, particularly includ-ing genetics, evolutionary biology, ecology, physi-ology, and microbial pathogenesis. The species has enabled scientists from many disciplines to gain profound insight into physiology of various organ-isms including humans. In fact, as of 2020, a total of six Nobel prizes have been awarded to scientists that conducted studies using D. melanogaster as a model organism. One of the most frequently exam-ined species over the last decades by biological and genetic research, as well as ecotoxicology, D. mela-nogaster could be the best-known eukaryotic organ-ism on planet Earth.
Although the toxicity of nanopesticides has been investigated in different model organisms, there is rather limited research to examine the impacts of nanopesticides using Drosophila as an experimental model. One relatively recent study, possibly the only one so far, investigated possible toxic effects of Ag NPs and sulfur (S) NPs on larval, pupal, and adults of D. melanogaster, reporting significantly high mortality and reduced longevity for both types of NPs (Araj et al. 2015). Kah and Hofmann broadly classified nano-based plant protection products into five groups according to their active ingredients, which include (1) nanoemulsions, (2) polymer-based nanopesticides, (3) hybrid nanoformulations, (4) inorganic NPs associated with an organic active ingredient, and (5) inorganic NPs as active ingredi-ent (Kah and Hofmann 2014), but Table 1 presents a more detailed categorization of current nanopesti-cides. Some of the NMs like Si, TiO2, Ag, Au, and Co, which are commonly used as active ingredients in nanopesticide formulations were already exam-ined in toxicity and genotoxicity studies using Drosophila. Therefore, reviewing such studies may provide crucial insight into the effects of nanopesti-cides. The first study to examine the impacts of NM (cerium oxide-CeO2 NPs) exposure on D.
mela-nogaster was carried out by Strawn, Cohen, and Rzigalinski (2006). Since then a multitude of research into genotoxicity, cytotoxicity, and biocom-patibility of various NMs of different shape, size,
and matter has been using Drosophila as a model organism. Although the current knowledge is rather limited to establish a clear picture of toxicity of NMs, as there are contradicting findings about their effects on ROS generation, DNA damage, reproduct-ive capacity, and viability, a significant portion has been demonstrated to cause toxic effects during in vivo testing on this model organism. For example, in their in vivo study, Demir (2020) found that exposure to TiO2 NPs triggered cellular uptake, toxicity, DNA damage, and oxidative stress in D. melanogaster. Besides, detrimental effects of Au NPs, Ag NPs, cobalt (Co) NPs and Si NPs on Drosophila, including somatic mutation, gene muta-tion, toxicity, impaired fertility and longevity, have been reported by several studies (Demir et al.2011; Vecchio et al. 2012; Armstrong et al. 2013; Pandey et al.2013; Vales et al.2013; Alaraby et al.2020).
The growing interest in utilizing Drosophila in toxicity studies ultimately led to emergence of a research field called Drosophotoxicology (Rand 2010). This new field involves a range of methodo-logical approaches using Drosophila as a model organism in toxicity and genotoxicity research (Chifiriuc et al. 2016). Toxicological assays designed to test NM exposure in Drosophila include compo-nents such as chemical toxicants, mode of delivery to the organism, developmental stage of the ner-vous system, and endpoints to be assessed for detecting biological and toxicological effects. In this context, the mode of delivery plays a crucial role in exposing the cells or organ systems in flies to NMs, and such modes may involve embryonic exposure through maternal feeding, delivery by direct injec-tion into embryo, and direct incubation of Drosophila embryos. Larvae and adult flies can be easily exposed to different concentrations of NMs through food ingestion, injection, and vapor/aerosol in a controlled environment. Acute and chronic tox-icity of NMs could then be assessed by means of a wide variety of assays designed to characterize sev-eral factors like survival, fecundity, DNA damage, morphological defects, and neurological health (Rand, Dao, and Clason 2009; Demir et al. 2011; Vales et al.2013; Alaraby et al.2020; Demir2020).
Since the whole human genome was first mapped and sequenced in 2003, comparison of complete-genome sequences of various species has demonstrated that humans share a substantial
number of genes with all other living organisms, including fruit flies. Comparative genomic research involving side-by-side analysis estimates that approximately 60% of Drosophila DNA is identical to that of humans, and almost 75% of the genes associated with human diseases, such as autism, diabetes, and cancer, have functional homologs in D. melanogaster (Lloyd and Taylor 2010). Drosophila has also been used as a simple and readily available genetic model organism by research into underly-ing mechanisms of immunity, agunderly-ing, oxidative stress, neurodegenerative disorders (Bier 2005), spi-nocerebellar ataxia (Latouche et al. 2007), and Alzheimer’s disease (Moloney et al.2010).
Another compelling feature of Drosophila is that it shares various basic biological and physiological mechanisms and molecular pathways with mam-mals (Pandey and Nichols 2011; Wang et al. 2012), which makes this insect an excellent model organ-ism for a wide range of fields, including pharmaco-logical research (Pandey and Nichols 2011), genotoxicity studies (Pandey and Nichols2011), and neurotoxicity screening (Rand 2010), where mam-malian model organisms are traditionally deemed an indispensable part of animal testing. This model organism partly owes its success to a series of advantages over vertebrate animal models, includ-ing rapid life cycle, ease of culturinclud-ing, low produc-tion costs, large offspring production per generation, high fecundity, and relatively simple genetics with only four pairs of chromosomes. Most importantly, ethical issues associated with verte-brate animals do not apply to fruit flies (Jennings 2011). Finally, the Drosophila toolbox that allows easy genetic manipulation is unprecedented among model systems (Mohr et al.2014). Besides these fea-tures, Drosophila is not only sensitive to the toxic effects of traditional pesticides but also it has been considered as a good model to test these pesticides associated with the use of resistant strains. Chemical pesticides constitute a vital tool in con-trolling most of the world’s destructive pests, yet utilization of such products in great amounts also cause pests to develop resistance, which leads to serious repercussions for sustainable pest control. In that regard, the mechanisms through which pests develop resistance should be well established. Such an effort involves identification and functional char-acterization of potential resistance genes or
mutations. Using non-target model organisms such as D. melanogaster, as well as recent advances in genome modification technology, most notably CRISPR/Cas9, has considerably accelerated research into mechanisms of pest resistance (Perry and Batterham 2018; Douris et al. 2020). Indeed, previ-ous work in the relevant literature contains hun-dreds of instances of pesticide resistance that is associated with variation in the overexpression of metabolic enzymes such as cytochrome P450s, glutathione-S-transferases, carboxylesterases, and UDP-glucuronosyltransferases. Therefore, overex-pression of metabolic genes from pests also found in D. melanogaster has been demonstrated to be a powerful tool for elucidating the link between pest resistance and enzyme activity (Ibrahim et al.2015).
Future prospects
Drosophila can be a highly valuable model in meet-ing today’s demands of toxicity studies into nano-based pesticides by allowing optimization of path-way-specific screening, facilitating rapid testing of samples for biological activity at cellular or molecu-lar levels, and rapid identification of genes respon-sible for interactions with NPs. Drosophila is an ideal model for high throughput screening as its fast generation time enables processing of thousands of flies for a given screen. Its ability to reflect the true interactions between DNA and environment might afford profound insights into the toxicity mecha-nisms of certain substances in humans – so much so that Collins, Gray, and Bucher (2008) has pro-posed a new model where Drosophila is expected to bring a paradigm shift in toxicity studies. Future efforts are geared toward better throughput screen-ing with a high degree of pathway specificity usscreen-ing in vivo assays based on Drosophila. Although in vivo toxicity studies carried out with alternative non-mammalian models like zebrafish have been claimed to reflect vertebrate response, Drosophila appears to be superior in many ways since it ena-bles researchers to process a large number of sam-ples and to identify antibodies and genes regulating certain pathways at much lower costs, thus granting access to valuable data on various parameters, including survival, mortality, longevity, mutagenic and recombinogenic activity (Demir
et al. 2011; Vales et al. 2013; Alaraby et al. 2020; Demir2020).
In conclusion, D. melanogaster might prove to be an ideal model organism for the risk assessment and toxicological classification of nanopesticides. Valuable contributions from the brand new research field known as Drosophotoxicology will help pro-vide better insight into possible impacts of nano-based pest control products, which are more com-plex by design, on the environment and human health. The ongoing collaboration between humans and Drosophila fruit fly since biologist Thomas Morgan first used it as a model organism for his research on heredity may once again help us unravel such complex biological and eco-logical processes.
Disclosure statement
The author reports no conflict of interest. The author alone is responsible for the content and writing of the paper.
ORCID
Es¸ref Demir http://orcid.org/0000-0002-2146-7385
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